1. Field of the Invention
The present invention relates to an animal model for human perimenopause and menopause. Also provided by the present invention are methods of making the animal model and methods of screening and using the model. Also provided are methods of inducing ovarian failure in animals such as pets and wildlife.
2. Description of the Background
The average age of menopause in women in the U.S. is 51 years. Demographic studies on the age of menopause have shown that it has increased from about 45 years in 1850, to approximately 51 in 1995. At the same time, however, the life expectancy of women has increased from 45 in 1850 to approximately 82 in 1998. As a result, because the life span in women has increased, almost 30% of a woman's lifetime will be postmenopausal (1). The consequence of this shift is that many age-related diseases are increasing in incidence and need to be investigated in relevant animal models to understand the effect of menopause on disease risk, presentation and progression. Many disorders such as Alzheimer's disease have an increased incidence in females of a relatively late onset (approximately 80 years of age). Obviously, in the future, the increase in life expectancy will impact the incidence of many age-related diseases and require aggressive intervention during the postmenopausal years.
Many health risks are known to be associated with menopause. There is a strong direct link between menopause and an increase in cardiovascular disease which is the leading cause of death in women over the age of 50 (2). A number of observational studies have, provided evidence that hormone replacement therapy (HRT) reduces the risk of cardiovascular disease by about one-half (3). However, the HERS study recently reported that HRT in post menopausal women did not prevent recurrent myocardial infarction (4). Recently, the Womens Health Initiative study conducted by the NIH reported a slight increase in cardiovascular disease-associated conditions in women taking HRT (5). Furthermore, there is a significant debate over the advantages and disadvantages of using HRT in postmenopausal women relative to a potential increase in both breast and ovarian cancer risks (5, 6, 7). Clearly, studies utilizing a relevant animal model would contribute greatly to resolving these issues. Interestingly, although controversial, it has been suggested that there are benefits rather than risks associated with the estrogens in birth-control pill usage in premenopausal women (7). These seemingly disparate effects of estrogen treatment will best be resolved in the laboratory.
Menopause is the cessation of ovarian cyclicity resulting from the depletion of ovarian follicles by a natural process of atrition, known as atresia (8). Follicular maturation in the ovary is a dynamic series of events in which primordial follicles provide a finite pool from which preovulatory follicles are selected for development and ovulation, or are eliminated by atresia. The primordial follicle, the most immature stage of development, is formed in the ovary during fetal development. Because the oocyte is arrested in meiosis, this pool is non-regenerating after birth. In an on-going process, after puberty, follicles continually progress from the primordial to ovulatory stages. However, the vast majority do not develop to ovulation, but undergo cell death by atresia. As a result, the pool of primordial follicles gradually becomes depleted and ultimately, ovarian failure (menopause) ensues (8). As the pool of primordial follicles is depleted, this compromises the numbers of developing preovulatory follicles. Eventually, the reduction in pre-ovulatory follicles significantly alters ovarian steroid hormone production as a woman approaches menopause (perimenopause), resulting in a sharp decrease in circulating 17β-estradiol and a concomitant rise in the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH), due to loss of negative feedback from the ovary to the pituitary. Thus, following menopause, hormonal cyclicity ceases within the ovary and it secretes primarily androgens in a hypergonadotropic environment (8). Because 17β-estradiol is assumed to afford protection in premenopausal women against health risks, such as cardiovascular disease, skeletal problems, and brain dysfunction, the loss of 17β-estradiol in menopause is thought to contribute to most menopause-associated disorders.
In researching menopause, a limited amount of mechanistic information can be obtained from studies in middle-aged women. Therefore, the elucidation of underlying cellular and molecular mechanisms that accompany menopause-associated disorders requires the use of appropriate animal models in controlled experimental conditions. Although nonhuman primates most closely resemble humans, there are disadvantages in using them in the study of menopause. These include limitations on the number of animals, costly acquisition and housing expenses, and lengthy life spans, with reproductive senescence occurring late in life (9). Rodent models, on the other hand, are inexpensive and reproductive senescence can be caused and studied within a relatively short time frame. Previous rodent studies have attempted to model menopause by ovariectomy. While this approach mimics the loss of 17β-estradiol seen in menopause, it lacks consideration of the physiological contributions of the postmenopausal ovary. It is possible that the postmenopausal ovary impacts the effects of the changing hormonal and gonadotropin milieu via secretion of bioregulatory factors. In postmenopausal women who have had their ovaries removed, a 50% reduction in testosterone has been observed, indicating that the senescent ovary is secreting androgens that have the potential to impact postmenopausal health risks (10). However, because there has not heretofore been an adequate animal model of the postmenopausal ovary, this issue has not been directly investigated. In surgical experiments conducted in mice, aged ovaries were transplanted into young ovex recipients. Plasma FSH levels were significantly increased compared to controls, indicating that the aged ovary plays a role in reproductive failure by impacting the hypothalamic-pituitary-ovarian axis, perhaps by as yet-unknown bioregulatory factors as well as loss of negative feedback due to a reduction in 17β-estradiol. Collectively, the lack of information about the follicle-depleted ovary supports the need for investigations into its function in vivo as well as in vitro to understand the effects on age-related diseases associated with menopause.
The occupational chemical VCD, the diepoxide metabolite of 4-vinylcyclohexene (VCH), causes selective loss of small preantral follicles in the ovaries of mice and rats (11-13). Compared to vehicle controls, VCD dosing of rats and mice for 12 days caused a significant reduction in the number of primordial and primary follicles (12). However, following 30 days of dosing there was also a significant reduction in the numbers of secondary follicles (11, 13) which was explained as a reduction in the pool of primordial follicles from which secondary follicles could be recruited at that time to develop into large antral follicles that produce 17β-estradiol. The targeting of primordial and primary follicles by VCD appears to be follicle stage-specific because no direct effects have been observed or measured in larger (secondary to antral) follicles, or other non-ovarian tissues as determined by necropsy, histopathology, plasma lipid profiles, and liver enzyme activity. Using a combination of molecular and cellular approaches in our studies in rats we have collected evidence that VCD-induced follicle loss is by acceleration of the normal rate of atresia. These studies have also demonstrated that alterations in apoptosis-associated intracellular pathways activated by VCD dosing are specific for small preantral follicles, as compared with large preantral follicles or liver. Atresia in the rodent ovary occurs via apoptosis, or programmed cell death, and is a normal process without necrosis-induced responses such as inflammation. Evidence of VCD-induced impending follicle loss was observed as an increase in numbers of unhealthy follicles in the treated group. The unhealthy appearance in VCD-treated ovaries is morphologically and ultrastructurally similar to unhealthy follicles undergoing natural atresia in controls (14-16). At the molecular level, VCD-accelerated atresia in small preantral follicles was identified because intracellular events associated with apoptosis were measured selectively in the targeted follicular population. These events include: A) increased Bax/BclxL ratio (17), B) increased expression of Bad, C) leakage of cytochrome c from mitochondria into the cytosol (18), D) increased caspase-3 activity (18), E) activation of the JNK and p38 branch of the MAPK signaling pathway (19), F) retardation of natural atresia and bax expression (relative to controls) in small follicles following a single dose of VCD (20) and G) estrogen receptor-mediated protection from VCD-induced follicle loss (21). Collectively, these data support at a molecular level that VCD causes follicle loss by enhancing events associated with the normal process of atresia. These molecular events are specific for the small preantral follicles known to be physiologically targeted by VCD.
Follicle loss resulting from repeated dosing of F344 rats with VCD has been well characterized (11-21). The conclusion from characterization performed in preliminary studies is that VCD causes the selective loss of primordial and primary follicles by accelerating the natural process of atresia via apoptosis. In long term studies in B6C3F1 mice (22) and F344 rats (15) that were dosed with the parent compound, 4-vinylcyclohexene (VCH, mice) or the ovotoxic form (VCD, rats), premature ovarian failure occurred within a year. However, the time it takes from dosing of the animals until premature ovarian failure occurs is too long in order to use animals prepared in such a manner as a model for menopause.
The development of an animal model that mimics the onset of menopause is critical to enhance an understanding of the role of menopause in many age-related diseases. By using VCD to chemically accelerate the normal process of atresia selectively in primordial and primary follicles, it would be possible to more accurately approximate the physiological events that occur during the progression from ovarian function through impending ovarian failure (perimenopause), to the eventual disease risks that result after menopause. Therefore, such a model would be powerful in scope because a wide variety of physiological and molecular endpoints can be designed for understanding the complexities of health risks that accompany menopause in women.